The overall goal of this proposal is to characterize the novel tumor suppressor activity of a key metabolic enzyme and to determine the mechanism(s) of its selective toxicity to malignant vs. non-malignant cells. FDH (10-formyltetrahydrofolate dehydrogenase) irreversibly converts 10-formyltetrahydrofolate, an essential substrate for de novo purine biosynthesis, to tetrahydrofolate. Through depletion of this substrate, FDH can restrict purine biosynthesis. In turn, this interferes with important downstream intracellular processes, including DNA/RNA biosynthesis and DNA repair. Because of this critical metabolic function, down-regulation of FDH in cancer cells was predicted to be prosurvival, while artificial elevation through stable transfection, would be toxic. We have recently made the important observation that FDH is strongly and ubiquitously down-regulated in tumor tissues and cancer cell lines and that FDH promoter hypermethylation is likely involved in this down-regulation. We have further demonstrated that moderate FDH expression in FDH-deficient cancer cells induces apoptotic cell death. Furthermore, evidence was recently obtained that p53 is required to mediate FDH-induced cytotoxicity. In contrast, non-cancer cells were insensitive to FDH elevation. Therefore, it is proposed that cancer cells silence the FDH gene in order to escape cytotoxicity. Our central hypothesis is that FDH down-regulation through promoter hypermethylation is one of the important means by which malignancies gain pro-survival advantage over normal cells. The Specific Aims that will be used to address this hypothesis are: (1) Determine the molecular mechanisms of FDH-induced apoptosis in transfected cancer cells, (2) Elucidate the mechanism(s) that protects non-malignant cells from FDH-induced apoptosis, and (3) Determine the role of promoter hypermethylation in down-regulation of FDH in cancer cells. Stable clones of malignant cells that inducibly express FDH, resistant cancer cell clones that have acquired the ability to constitutively express FDH, and FDH-insensitive non-malignant cells, will be used to pursue the goals of this project. It is proposed that investigation of the critical role of FDH in cancer cell survival will provide important insight into the malignant process itself and link disregulation of important metabolic pathways to cell death.